ELEMENTS CAUSING MALE CROSSING OVER IN DROSOPHILA MELANOGASTERI BARTON E. SLATKO
AND
Department of Zoology, The University
YUICHIRO HIRAIZUMI of
Texas at Austin, Austin, Texas 78712
Manuscript received January 13,1975 Revised copy received June 1,1975 ABSTRACT
A second chromosome line of Drosophila melanogaster (Symbol: T-007) has previously been shown to be responsible for the induction of male recombination. I n the present investigation, the genetic elements responsible for this phenomenon have been partially identified and mapped. A major element (Symbol: Mr, for Male recombination) locates on the second chromosome between the pr (2L-54.4) and c (2R-75.5) loci and is responsible for the large majority of male recombination. In addition, there appear to be “secondary elements” present which have the ability to induce male recombination i n much reduced frequencies and which are diluted out through successive backcross generations when M r is removed by recombination. The possible nature of these “secondary elements” is discussed.
as been generally assumed that crossing over is absent in Drosophila males, ‘;etpite the fact that only a relatively small number of species have been adequately tested. Notable exceptions to the aforementioned assumption include certain strains of Drosophila ananassae (KIKKAWA1937; HINTON 1970; MORIWAKI and TOBARI 1973) and Drosophila willistoni (FRANCA, DA CUNHAand GARRIDO 1968). MORGAN first reported the absence of crossing over in male Drosophila melano1912, 1914), and this observation has since been confirmed by gaster (MORGAN numerous investigators. It was not until recently that a natural population of Drosophila melanogaster was found to contain genetic elements that could give 1971; HIRAIZUMI et al. 1973). They rise to male recombination (HIRAIZUMI reported that a large percentage of the second chromosome lines isolated from a natural population in southern Texas were able to undergo male recombination, although in frequencies much lower than those in females. Since this first report, a number of other investigators have observed similar phenomena in Drosophila melamgaster isolated from natural populations in numerous geographical locations including: Florida (VOELKER 1974), North Carolina (BROADWATER et al. 1973), Ohio (WADDLE and OSTER1974), Oklahoma (WOODRUFF and THoMP~oN, personal communication), Pennsylvania (YAMAGUCHI and MUKAI1974),several New England populations maintained in the laboratory for less than 10 years (KIDWELLand KIDWELL1975), Cambridge, England (WOODRUFF, personal communication),and Australia (SVED1974). 1
This work was supported by research grant NIH GM-19710.
Genetics 81: 313-324 October, 1975.
314
B. E. SLATKO A N D Y. HIRAIZUMI
In addition to the induction of male recombination, males heterozygous for the second chromosomes, isolated by HIRAIZUMI, were found to show segregation frequencies from heterozygous male parents much lower than the Mendelian expectation of 0.50. SLATKO and HIRAIZUMI (1973) further showed that these chromosome lines acted as "mutator strains", as evidenced by increased frequencies of X and second chromosome recessive lethals, visible mutations, and mosaics. One conspicuous absence from previous studies concerned the identity of the genetic elements responsible for the induction of the male recombination phenomenon. The purpose of the present study is to report on this aspect. MATERIALS A N D METHODS
Strains of Drosophila melanogaster used for the present study are listed as follows. 1. cn bw: A standard second chromosome line marked with two recessive eye color mutants, cn (cinnabar eye color, 2R-57.5) and bw (brown eye color, 2R-104.5). Homozygous flies, cn bw/cn bw, show white eye color. 2. Zn(ZLf2R) Cy; cnz bw: A second chromosome line with two large inversions, one in the left and the other in the right arm. This chromosome carries the dominant marker C y (Curly wing) and two recessive eye color mutants, cne (an allele of cn) and bw. Homozygous lethal, this line will be abbreviated as Cy. 3. a1 d p b p r c p z s p : A second chromosome line marked with seven recessive markers, al (aristaless, 2L-0.01), d p (dumpy wing, 2L-13.0), b (black body color, &L-48.0),p r (purple eye color, 2L-54.4), c (curved wing, 2R-75.5), p z (plexus wing vein, 2R-la0.5), and s p (speck, 2R-107.0). This line will be abbreviated as apl in this report. 4. T-007: One of the second chromosome lines that showed male recombination in the frequency of about 1% between cn and bw. This line was isolated from a natural population in Harlingen, Texas. Males collected from the natural population were first mated to C ( l ) D X , y f ; cn bw females (to replace their Y chromosomes) and progeny males were then backcrossed to cn bw females for nine generations before being kept as a balanced stock, T-OM/Cy. T-007 is associated with recessive lethality. A standard cornmeal-agar food, supplemented with proprionic acid as a mold inhibitor, was used throughout these experiments. The age of all flies at the time of matings was usually less than six days. Parents were kept in a vial for seven days and then discarded. Progeny were counted through the nineteenth day from the date of mating. All experiments were carried out at room temperature, 23"-24". EXPERIMENTS A N D RESULTS
Mapping of the elements causing male recombination: The T-007 second chromosomal line has been kept in o w laboratory as a balanced stock after many generations of backcross matings (see MATERIALS AND METHODS) which selected only for the second chromosome. It is apparent that at least one major element causing male recombination must locate on the second chromosome, as all other chromosomes would have been diluted out during the course of the backcross matings initiated in 1971. The second chromosome of this line was thus examined in order to map the position of the elements responsible for male recombination. The mating scheme used for this purpose is shown in Figure 1(A). Initially, T-O07/Cy males were mated to apl females. From this mating, virgin F, T-007-apl females were selected and backcrossed to homozygous apl males. Among the progeny of these matings were individuals bearing various recombi-
M A L E RECOMBINATION IN DROSOPHILA
315
B
A
R
cnbw 88 G,,
1 F4
R d cn bw
cn bw 89
FLY COUNTS EXPERIMENT 1
cn bw 98 G4
cn bw
I
FLY COUNTS EXPERIMENT 1
FIGURE 1.-A. Mating scheme utilized to isolate recombinant chromosome types (R) generated between the apl and T-007second chromosomes, including isolation of the presumptive nonrecombinant chromosomes of the genotype and to subsequently test them for the ability to induce male recombination between the second chromosome right arm markers cn and bw. B. Mating scheme utilized to isolate the presumably non-recombinant chromosomes of the a2 d p b pr c pz sp genotype, and to subsequently test them for the ability to induce male recombination between cn and bw.For full details consult text.
+ + + + + + +,
nant chromosomes which were generated in the heterozygous F, females. These recombinant chromosomes were then assayed for the presence or absence of the ability to induce male recombination between the two recessive markers cn and bw. This was done as follows: The F, males of the appropriate recombinant genotypes were individually mated to cn bw females, and from each of these matings, F, males were isolated. All F, males collected from each Fzmating were phenotypically “wild type” but genotypically either apl/cn bw or recombinant chromosome type/cn bw. It was therefore necessary to double-mate individual F, male
316
B. E. SLATKO A N D Y. HIRAIZUMI
progeny from each original F, recombinant male first to apZ females (FZain Figure 1( A ) ) to select males of the appropriate genotypes and then to cn bw females (Fa,,in Figure 1(A)) to isolate a larger number of recombinant chromosome type/cn bw males. The F4males were genotypically heterozygous f o r cn bw and the identical “recombinant chromosome” present in the F, parental males. These heterozygous males were then mated to cn bw females to test f o r the presence of the ability to induce male recombination. By this scheme, each of the individually isolated recombinant genotypes could be tested for the ability to induce male recombination between cn and bw, and in addition, it was possible to obtain a fairly large number of F, males (i.e., replications) for each individually isolated recombinant chromosome. It should be noted that from this mating scheme it is also possible to isolate the genotype presumptive non-recombinant chromosomes of the (= T-007),but impossible to isolate the non-recombinant apl chromosomes. This is due to the fact that in the F, matings in Figure 1(A), it is impossible to distinguish the apZ chromosome derived from the F, females and the apZ chromosome from F, males. For the purpose of isolating the non-recombinant apt chromosomes, an alternative mating scheme was used, which is shomwn in Figure 1 (B) . The results of these matings, designated as Experiment 1, are summarized in Table 1. Table 1 includes the results obtained for the 12recombinant and 2 presumptive non-recombinant genotypes (Type numbers 1-14) generated in the T-O07/apZ heterozygous females, which were tested for the ability to induce male recombi-
++ ++ +++
TABLE 1 Male recombination frequencies between the cn and bw loci for the twelve recombinant and two presumptive non-recombinant chromosome types generated in T-O07/apl heterozygous females TJ-w
Group number
A
1 2 3 4
B
5 6 7 8 9 10 11
c
12 13 14
N2+
Avg. no. replic. per line
Avg. no.
NI*
progeny per line
RE. h-eq. between cn and bw
9 6
0 0 0 0
19 4 9 6
28.5 31.5 21.3 32.2
1682 1727 1399 1874
0.0397 0.0100 0.0079 0.0398
aZdpbpr+++ a l d p b p r c + + a1 d p b p r c px a1 dp b p r c px sp dp b pr c px sp b pr c p x s p pr c P X SP
10 6 5 21 4 9 7
6 3 2 9 0 2 0
4 3 3 12 4 7 7
43.5 25.0 18.0 24.4 27.6
2235 1323 2199 1612 1330 1930 1585
0.0018 0.0005 0.0012 0.0009 0.0019 0.0020 0.0025
+ + + + + + S P
11 10 4
1 0 0
10 10 4
41.2 22.5 25.5
2187 1471 1393
0.0061 0.0063 0.0102
iTotal no. of lines isolated
Recombinant chromosome type
+++++++ al++++++ aldp+++++ a l d p b + + + +
19 4
+
+ ++ +++ f + + + c px s p $. + -k + + p x s p
19.2 44.0
~~
* Nupber of lines which did not induce any male recombination.
-f Number of lines which induced some male recombination. For further details, consult text.
MALE RECOMBINATION I N DROSOPHILA
317
nation between the second chromosome right arm markers cn and bw. For each recombinant chromosome type isolated (F, or Gzmales in Figure 1) ,a number of independently isolated chromosome lines were collected (designated as “Total number of lines isolated” in Table 1). For each independently isolated chromosome line, a number of replications were made (corresponding to the F4o r G4 males, Figure 1). The average number of replications and the average number of subsequent progeny scored for each chromosome line are shown for each recombinant chromosome genotype. Additionally, the number of independently isolated recombinant chromosome lines showing male recombination induction are listed, along with the recombination frequency (measured between cn and bw) averaged for all lines of each recombinant chromosome type. For example, in Group B, 21 independent (F,) males with the a l d p b p r c p x s p genotype (type number 8) were isolated. For each of the 21 lines, the number of replications (F, males) averaged 25.0 and the number of progeny (averaged for the 21 lines) was 1612. Of the 21 lines, 12 induced some male recombination. The recombination frequency between cn and bw, averaged for all 21 lines, was 0.0009 (almost 0.1 %). Because the phenomenon of male recombination seems to be of largely premeiotic origin (HIRAIZUMI et al. 1973), any computations of average recombination frequencies must take into consideration the occurrence of clustering. Although a deviation in the distribution of recombinants among males from the Poisson expectation was statistically significant, the degree of clustering was generally small. In addition there were no large differences, with respect to clustering, between the 14 recombinant chromosome types. For all practical purposes, therefore, the use of a simple average recombination frequency is permissible, neglecting the small clustering effect. The recombinant chromosome types listed in Table 1 have been organized into three groups (Groups A to C) . It can be seen that with respect to the ability to induce male recombination, the three groups can be classified into two sets. The first set (Groups A and C) induces a relatively higher frequency of male recombination than the second set (Group B) . The recombinant types in the first set (Groups A and C) have only one region in common: the pr+ region derived from the T-007 chromosome. Conversely, those recombinant chromosome types in the second set contain the pr region from the apl chromosome. Note that of a total of 63 chromosomes carrying the pr+ region which were tested, only one (of the type + + + + c p x s p ) did not induce any male recombination. All of these observations seem to suggest that one major element responsible for the induction of male recombination is located near, and probably to the right of, the pr+ locus in the T-007 chromosome. As was mentioned previously, practically all (i.e., 62 out of 63) of the recombinant chromosomes carrying the pr+ region from T-007 induced male recombination. If one assumes that only a single element is responsible for male recombination induction, the majority of the complementary recombinant chromosome types (which carry the pr region from the apl chromosome) should not induce male recombination. However, this does not appear to be the case; the
318
B. E. SLATKO A N D Y . HIRAIZUMI
majority (40 out of 58) show the induction of male recombination, although in markedly reduced frequencies. These results suggest that in addition to the major element located near the pr+ region of the T-007 chromosome, there must be at least one additional “secondary element” present which is also responsible for the induction of male recombination, If a single “secondary element” which can induce low levels of male recombination is assumed, then by examination of the lines in Group B (lines not carrying pr+ from T-007), it should be possible to’ localize this element. The assumption of a single second locus would necessarily imply the existence of two distinct classes of male recombination in this group. It might appear, by preliminary inspection, that a “secondary element” may be present around the al+ locus, since 18 out of the 20 recombinant chromosome types with al+ (types 9, IO, and 11) showed some male recombination. It can also be seen, however, that this single “secondary element” model does not entirely explain the observed results, since more than half (22 out of 42) of the remaining recombinant types (types 5 , 6, 7, and 8) also show some male recombination induction. One other interesting observation from the data in Table 1 that pertains to the analysis of the “secondary elements” concerns the presumptive non-recombinant chromosome of the genotype a1 d p b pr c p x sp. Flies of this type carry all seven mutant markers. and therefore have the same genotype as the standard apl chromosome, except f o r the possible occasional occurrence of double crossover events (in the T-O07/apl females) between any two adjacent markers. Also, possible single crossover events outside of the a1 and sp markers (to the left of a1 or to the right of sp) might occur such that the tip of the left or right arm of T-007 could be transferred to the “non-recombinant” chromosome. The frequency of such undetected double crossovers should be very low, as should be the frequency of recombination outside the two end markers, considering their map positions relative to the tips of the chromosomme arms. A total of 21 a1 d p b pr c px sp “nonrecombinant” chromosomes were tested for the ability to induce male recombination, as shown in Table 1. Of the 21 lines tested. 12 showed the ability to induce male recombination. This frequency (60%) is too high to be explained by the occurrence of double crossing over between any two adjacent markers, or by single crossover events outside the end markers (a1 and sp) . It should be stated here that neither the apl chromosome nor the cn bw chromosome induces any male recombination. From the mating apl/cn bw 8 X cn bw 0 , 7656 progeny were scored and no recombinants were observed. Similarly, from the mating apl/cn bw 8 x apl 0,4471 progeny were scored, and again, no recombinants were found. Thus, the c n bw chromosome and the apl chromosome do not contain genetic elements which can cause male recombination. These results suggest that in addition to the presence of the major locus f o r male recombination induction, ‘Lsecondaryelements” must be present which can induce low levels of male recombination. Since the T-007 second chromosomal line was originally kept by repeated backcross matings to cnbw, only the originally isolated second chromosome remains in the T-O07/Cy stock. Any genetic elements located on any other chromosome would have been diluted out.
319
M A L E R E C O M B I N A T I O N IN DROSOPHILA
Thus, there must be an association between the “secondary elements” and the T-007 second chromosome. We will return to reconsider these “secondary elements” later. Many of the chromosome lines included in Table 1 were discarded after the completion of Experiment 1. Several of the lines, however, were kept by repeated backcross matings through males to cn bw females. After six generations of these backcross matings, these lines were re-examined for the ability to induce male recombination. The results of these matings, designated as Experiment 2, are shown in Table 2. Experiment 2 was originally designed to obtain some confirmation of the results presented in Table 1. The results, however, provided much more important information than a mere confirmation. Whereas the data presented in Table 1 is a summary of all independently isolated chromosome lines of a given genoTABLE 2 Comparisons of male recombination frequencies for several recombinant chromosome types between Experiment 1 and Experiment 2 Experiment 1
Group
A
Recombinant chromosome type
+++++++ ++ ++ ++ ++ ++ ++ ++ +++++++ -
Line number
50 50 47 47
2421 2690 1964 2298
0.0050 3.0126 0.0041 0.0083
20 17 20 19
935 858 1087 926
0.0011 0.0163 0.0055 0.0088
al++++++ al++++++
3 4
48 43
2175 2388
0.0092 0.0101
14 20
739 1184
0.0149 0.0042
aldp b dp b
2
44 47
2109 2532
0.0079 0.0122
20 20
1271 1306
0.0047 0.0100
++ ++ ++ ++
5
0.0087
al d p b pr c
++
a1 d p b p r c px s p
a1 d p b pr c p x s p
a1 a1 a1 a1
-
dp dp dp dp
b b b b
0.0082
4
20
890
0.0000
20
1267
0.0000
8 9
17
20
942 980
0.0000 0.0000
20 20
1214 1333
0.3000 0.0000
o.cooo
Mean
c
Experiment 2
No. No. Rec. freq. males progeny between tested scored cn and bw
8 9 11 13
Mean
B
-
No. Rac. freq. No. F male: progeny between tested scored cn and bw
c px s p c px sp
0.0000
7 10 11 12
23 47 20 40
1007 2424 909 1749
0.0020 0.0017 0.0022 0.0017
30 30 29 30
2033 2093 1741 2102
0.0000 0.0000 0.0000 0.0000
3 4
23 20
942 920
0.032 0.0011
40
44
2694 2410
0.0000
p r c P X SP
P r c PZ S P
3
48
2275
0.0304
40
2874
0.0000
pr pr pr pr
+ dP b ‘P + 4~b +++
c px s p c px s p
c
PZ SP
Mean
For full details, consult text.
0.0018
0,0000
0.0000
320
B. E. SLATKO A N D Y. H I R A I Z U M I
type, the data in Table 2 is presented so that for each individual line of a given genotype, direct comparisons can be made between the results oibtained in Experiment 2 (after 6 generations of backcross matings) . Before examining the results presented in Table 2, one aspect which first should be considered concerns any change in the “recombinant chromosome genotype” due to male recombination which could have occurred during the s i x generations of backcross matings to the cn bw females. Any recombination between the cn and the bw loci could be detected by phenotype. In fact, such recombinants were occasionally found and eliminated during the stock-keeping procedures. Since the cn bw chromosome does not carry any markers in the left arm, male recombination in the left arm could not be detected in this manner. Before experiment 2 was performed, a check was made for the presence of the left arm markers characteristic for each of the “recombinant chromosome” lines. This screening was done by mating the heterozygous males, (recombinant chromosome type /en bw), to apl females and examining the phenotypes of the progeny flies. This works efficiently for the lines carry the left arm end marker, al, but does not work for lines such as c px sp, which lack left arm markers. Consequently, there is the small possibility of undetected male recombination in the left a n n of such genotypes. In addition, there is no way to detect any double crossover events which might have occurred between any two adjacent markers, or single crossover events outside of the two end markers, al, and sp. The possibility of such undetected male recombination should be very small, however, since the frequency of male recombination is generally low. Based upon these considerations, it seems probable that a large majority (presumably all) of the “recombinant chromosome” lines tested in Experiment 2 still maintained the same genotypes originally present when they were tested in Experiment 1. In Table 2, the recombinant chromosome types are divided into three groups (Groups A, B, and C) . Group A includes those lines which carry the pr’f region from T-007 and which induced relatively higher frequencies of male recombination in Experiment 1 (Mean = 0.0087). When retested after six generations of backcross matings, these lines continued to induce male recombination in comparable frequencies (Mean = 0.0082). As mentioned previously, several lines tested in Experiment 1 failed to induce male recombination. With only one exception, these were restricted to lines which carry the pr region from the apl chromosome. Group B in Table 2 lists such lines, i.e., those lines which induced no male recombination in Experiment 1 and which were retested in Experiment 2. It can be seen that those lines which were unable to induce male recombination in Experiment 1 remained unable to do so when retested after the six backcross generations. The most interesting observation from the date presented in Table 2 concerns those lines which are inchded in Group C. These are the lines which carry the pr region from the up2 chromosome and which showed relatively lower frequencies of male recombination in Experiment 1. When these lines were retested after six generations of backcross matings, none was able to induce male recombination any longer. In these genotypes, the ability to induce male recombination is now very much reduced.
+ +++
321
MALE RECOMBINATION I N DROSOPHILA
Because there was a previous suggestion that the “major element” responsible for the induction of male recombination locates between the pr+ and c+ loci in the T-007 chromosome, the two complementary recombinant chromosome types generated between these two markers (i.e., c px s p and a2 d p b p r +) were examined separately from the recombinant types listed in Table 2. The results from the matings designed to retest these two recombinant types for the ability to induce male recombination after the six generations of backcross matings are shown in Table 3. Table 3 is analogous to Table 2 (in design and intention), with the exception that only the two complementary recombinant chromosome types, c p x sp and a2 d p b pr are considered. Group A includes those lines which induced relatively higher frequencies of male recombination in Experiment 1. It can be seen that these lines have retained the ability TO induce male recombination when retested after six generations of backcross matings. It is interesting to note that both of the complementary genotypes are represented in this group, as expected if the “major element” locates between the p i + and c+ regions of the T-007 chromosome. Group B in Table 3 includes those lines which induced no recombination in Experiment 1. The lines included in this group are still unable to induce male recombination, analogous to the data presented for Group B in Table 2. Group C in Table 3 contains a single representative. This line induced relatively lower male recombination in Experiment 1; but when retested after the 6 generations of backcross matings, this line was found to be unable to induce
+ +++
++
++++
+ + +,
TABLE 3
+++
Comparisons of male recombination frequencies for the a1 dp b pr and px sp recombinant chromosome types between Experiment I and Experiment 2
+ + + +c
~
~
~~~
Expenment 1
No F4 Group
A
Recombinant chromosome type
++++ ++ ++ ++ ++ a1 dp b pr a1 dP b Pr
c PXSP c PXSP
c PZSP
++ ++ ++
Line number
No.
Rec. freq. males progeny between tested scored cn and bw
~-
5 6 7
50 37 47
2266 2491 2975
4 10
20 53
1960 2716
++ ++ ++ a l d p b p r + + + al dP b Pr + + + dp b pr
2 3 8 9
45 50 40 40
2104 2328 2630 2390
a l d p b p r + + +
For full details, consult text.
1
21
1900
Dec. f q .
0.006.E
20 20
1063 1336 1348
0.0019 0.0112 0.0052
0.0034 0.0056
40 30
2557 1972
0.0023 0.0076
0.0013 0.3129
20
0.0000 0.0000 0.0000 0.0000
0.0056 30 20 20
20
1819 1142 1330 1477
0.0000
Mean
c
No.
males progeny between tested scored cn and bw
0.0059
Mean al dP b Pr
Experiment 2
No.
0.0046
0.0000 0.0000 0.0300 0.0000
0.0000 30
1660
0.0000
322
B. E. SLATKO A N D Y. H I R A I Z U M I
male recombination any longer. This line thus acts similarly to those lines included in Group C in Table 2. These results seem to indicate that the maintenance of the ability to induce male recombination is due to a single element located between pr+ and C+ in the T-007 chromosome, as some of the lines of the genotype a1 d p b pr 4-4- still maintain the ability to induce male recombination in Experiment 2, and thus still carry the “major element”. This element may locate nearer to pr+ in the T-007 chromosome, although the number of lines tested is too few to determine its position accurately. When a chromosome carries this element, to which we give the name Mr (for Male recombination), this genotype has the ability to exhibit similar levels of male recombination through successive generations. AS was pointed out previously, M r is not the only element; there seem to be other “secondary elements” present which can induce male recombination in much reduced frequencies. Results of Experiment 2 indicate that the action of such “secondary elements” is rather transient, inducing male recombination f o r only a few generations.
+
DISCUSSION
The male recombination system in Drosophila melanogaster appears to be a complex system in which one major element, Mr, is responsible for the large majority of male recombination. Mr locates between the p r f and c+ loci in the T-007 chromosome, possibly nearer to the pr+ locus. In addition to this major element, there seem to be other “secondary elements” present whose abilities to induce male recombination are much less than that of Mr and whose effects are “diluted O U ~ ” through successive backcross generations. It is obvious that the elements responsible for male recombination must be associated with the second chromosome. However, this does not preclude the fact that this strain of Drosophila melanogaster could have had elements located on other chromosomes. Because the original isolation selected only the second chromosome, the influence of the other chromosomes is unknown. In fact, VOELKER (1974) and WADDLE and OSTER(1974) have obtained results that indicate that when both second and third chromosomes are extracted simultaneously from males isolated from “natural populations”, a higher frequency of male recombination is observed than if just the original second chromosome or the original third chromosome is present. At the present time, little data is available concerning the nature of the transitory “secondary element” effect. It was mentioned previously that neither the apl chromosome nor the cn bw chromosome induces male recombination (apl/cn bw males x cn bw females-7656 progeny, 0 recombinants; upl/cn bw males x up1 females-471 progeny, 0 recombinants). Thus, it is unlikely that either one of these stocks carries M r or any “secondary elements” that can induce male recombination. It is possible to envision that during backcrossing to c n bw,modifiers (which elements”) from the apl stock were being could be interpreted as LLsecondary diluted out. This would imply, however, that the apl stock must behave differ-
MALE RECOMBINATION IN DROSOPHILA
323
ently than the cn bw stack. Neither of these stocks induces male recombination, nor does either of these stocks show any type of suppression o r enhancement phenomenon. It is reasonable to conclude that the loss of “modifiers” (originally present in the apl stock) or the gain of “suppressors” (originally present in the cn bw stock) are not likely explanations for the evanescent “secondary element” effect. Ruling out these possibilities, the simplest explanation for this phenomenon rests with the T-007 genotype itself. It is interesting to note that this temporal “secondary element” effect could be the explanation for the speculations concerning transposable elements (HIRAIZUMI et al. 1973; KIDWELLand KIDWELL 1973; VOELKER 1974; WADDLE and OSTER1974) that have been advanced in order to explain some unusual aspects of this system. A number of other possible speculations concerning the transitory “secondary element” effect can be made. This phenomenon could perhaps most easily be explained by assuming that the “secondary elements” are products of M r ; however, one must consider the fact that the temporal effects are manifest for several generations. Obviously, a simple protein product of M r is an unlikely candidate. In fact, there is no a priori reason to assume that the “secondary elements” are physical particles. M r may induce some change in its homolog, which is temporal. This is similar in a number or respects to the paramutation phenomenon seen in a number of organisms (for recent review, see BRINK1973) and/or ribosomal DNA “magnification” seen in Drosophila melanogaster (RITOSSA 1968; TARTOF 1974). It is our feeling that a number of the previously mentioned speculations are experimentally testable. Investigations into this aspect of the male recombination system are the subjects for future studies. LITERATURE CITED
BRINK,R. A., 1973 Paramutation. Ann. Rev. Genet. 7: 129-150. BROADWATER, C., L. V. OWENS,R. PARKS,C. WINFREY and F. R. WADDLE,1973 Male recombination from natural populations of Drosophila melanogaster from North Carolina. Drosophila Inform. Serv. 50: 99. FRANCA, Z. M., A. B. DA CUNHA and M. C. GARRIDO, 1968 Recombination in Drosophila willisloni. Heredity 23: 199-204. HINTON,C. W., 1970 Identification of two loci controlling crossing over in males of Drosophila ananassae. Genetics 66:663-676. HIRAIZUMI, Y., 1371 Spontaneous recombination in Drosophila melanogaster males. Proc. Natl. Acad. Sci. US. 68:268-270. HIRAIZUMI, Y., B.SLATKO, C. LANGLEY and A. NILL, 1973 Recombination in Drosophila melanogaster male. Genetics 73 : 439-444.
M. G. and J. F. KIDWELL, 1975 Cytoplasm-chromosome interactions in Drosophila KIDWELL, melanogaster. Nature 235 : 755-756. KIKKAWA, H., 1937 Spontaneous crossing over in the male of Drosophila ananassae. 2301. Mag. 49: 159-160.
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B. E. SLATKO A N D Y. HIRAIZUMI
MORGAN, T. H., 1912 Complete linkage in the second chromosome of the male of Drosophila. 1914 No crossing over in the males of Drosophila of genes in Science 36: 710-720. -, the second and third pairs of chromosomes. Biol. Bull. 26: 195-204. MORIWAKI, D. and Y. N. TOBARI, 1973 Spontaneous male crossing-over of frequent occurrence in Drosophila ananassue from southeast Asian populations. Japan. J. Genetics 48: 167-173. RITOSSA,F., 1968 Unstable redundancy of genes for ribosomal RNA. Proc. Natl. Acad. Sci. U.S. 60: 509412. SLATKO,B. and Y. HIRAIZUMI, 1973 Mutation induction in the male recombination strains of Drosophila melanogaster. Genetics 75 : 643-649. SVED,J. A., 1974 Association between male recombination and rapid mutational changes in Drosophila mehanogaster. Genetics 77 (suppl.) : 64. TARTOF, K. D., 1974 Unequal mitotic sister chromatid exchange and disproportionate replication as mechanisms regulating ribosomal RNA gene redundancy. Cold Spring Harbor Symp. Quant. Biol. 38: 491-500. VOELKER, R. A., 1974 The genetic and cytology of a mutator factor in Drosophila melanogaster. Mutation Res. 22 : 265-276.
WADDLE, F. R. and I. I. OSTER,1974 Autosomal recombination in males of Drosophila mehnogaster caused by a transmissible factor. J. Genet. 61: 177-183. YAMAGUCHI, 0. and T. MUKAI,1974 Variation OF spontaneous occurrence rates of chromosomal aberrations in the second chromosomes of Drosophila melanogaster. Genetics 78: 1209-1221. Corresponding editor: A. CHOVNICK
AND
Department of Zoology, The University
YUICHIRO HIRAIZUMI of
Texas at Austin, Austin, Texas 78712
Manuscript received January 13,1975 Revised copy received June 1,1975 ABSTRACT
A second chromosome line of Drosophila melanogaster (Symbol: T-007) has previously been shown to be responsible for the induction of male recombination. I n the present investigation, the genetic elements responsible for this phenomenon have been partially identified and mapped. A major element (Symbol: Mr, for Male recombination) locates on the second chromosome between the pr (2L-54.4) and c (2R-75.5) loci and is responsible for the large majority of male recombination. In addition, there appear to be “secondary elements” present which have the ability to induce male recombination i n much reduced frequencies and which are diluted out through successive backcross generations when M r is removed by recombination. The possible nature of these “secondary elements” is discussed.
as been generally assumed that crossing over is absent in Drosophila males, ‘;etpite the fact that only a relatively small number of species have been adequately tested. Notable exceptions to the aforementioned assumption include certain strains of Drosophila ananassae (KIKKAWA1937; HINTON 1970; MORIWAKI and TOBARI 1973) and Drosophila willistoni (FRANCA, DA CUNHAand GARRIDO 1968). MORGAN first reported the absence of crossing over in male Drosophila melano1912, 1914), and this observation has since been confirmed by gaster (MORGAN numerous investigators. It was not until recently that a natural population of Drosophila melanogaster was found to contain genetic elements that could give 1971; HIRAIZUMI et al. 1973). They rise to male recombination (HIRAIZUMI reported that a large percentage of the second chromosome lines isolated from a natural population in southern Texas were able to undergo male recombination, although in frequencies much lower than those in females. Since this first report, a number of other investigators have observed similar phenomena in Drosophila melamgaster isolated from natural populations in numerous geographical locations including: Florida (VOELKER 1974), North Carolina (BROADWATER et al. 1973), Ohio (WADDLE and OSTER1974), Oklahoma (WOODRUFF and THoMP~oN, personal communication), Pennsylvania (YAMAGUCHI and MUKAI1974),several New England populations maintained in the laboratory for less than 10 years (KIDWELLand KIDWELL1975), Cambridge, England (WOODRUFF, personal communication),and Australia (SVED1974). 1
This work was supported by research grant NIH GM-19710.
Genetics 81: 313-324 October, 1975.
314
B. E. SLATKO A N D Y. HIRAIZUMI
In addition to the induction of male recombination, males heterozygous for the second chromosomes, isolated by HIRAIZUMI, were found to show segregation frequencies from heterozygous male parents much lower than the Mendelian expectation of 0.50. SLATKO and HIRAIZUMI (1973) further showed that these chromosome lines acted as "mutator strains", as evidenced by increased frequencies of X and second chromosome recessive lethals, visible mutations, and mosaics. One conspicuous absence from previous studies concerned the identity of the genetic elements responsible for the induction of the male recombination phenomenon. The purpose of the present study is to report on this aspect. MATERIALS A N D METHODS
Strains of Drosophila melanogaster used for the present study are listed as follows. 1. cn bw: A standard second chromosome line marked with two recessive eye color mutants, cn (cinnabar eye color, 2R-57.5) and bw (brown eye color, 2R-104.5). Homozygous flies, cn bw/cn bw, show white eye color. 2. Zn(ZLf2R) Cy; cnz bw: A second chromosome line with two large inversions, one in the left and the other in the right arm. This chromosome carries the dominant marker C y (Curly wing) and two recessive eye color mutants, cne (an allele of cn) and bw. Homozygous lethal, this line will be abbreviated as Cy. 3. a1 d p b p r c p z s p : A second chromosome line marked with seven recessive markers, al (aristaless, 2L-0.01), d p (dumpy wing, 2L-13.0), b (black body color, &L-48.0),p r (purple eye color, 2L-54.4), c (curved wing, 2R-75.5), p z (plexus wing vein, 2R-la0.5), and s p (speck, 2R-107.0). This line will be abbreviated as apl in this report. 4. T-007: One of the second chromosome lines that showed male recombination in the frequency of about 1% between cn and bw. This line was isolated from a natural population in Harlingen, Texas. Males collected from the natural population were first mated to C ( l ) D X , y f ; cn bw females (to replace their Y chromosomes) and progeny males were then backcrossed to cn bw females for nine generations before being kept as a balanced stock, T-OM/Cy. T-007 is associated with recessive lethality. A standard cornmeal-agar food, supplemented with proprionic acid as a mold inhibitor, was used throughout these experiments. The age of all flies at the time of matings was usually less than six days. Parents were kept in a vial for seven days and then discarded. Progeny were counted through the nineteenth day from the date of mating. All experiments were carried out at room temperature, 23"-24". EXPERIMENTS A N D RESULTS
Mapping of the elements causing male recombination: The T-007 second chromosomal line has been kept in o w laboratory as a balanced stock after many generations of backcross matings (see MATERIALS AND METHODS) which selected only for the second chromosome. It is apparent that at least one major element causing male recombination must locate on the second chromosome, as all other chromosomes would have been diluted out during the course of the backcross matings initiated in 1971. The second chromosome of this line was thus examined in order to map the position of the elements responsible for male recombination. The mating scheme used for this purpose is shown in Figure 1(A). Initially, T-O07/Cy males were mated to apl females. From this mating, virgin F, T-007-apl females were selected and backcrossed to homozygous apl males. Among the progeny of these matings were individuals bearing various recombi-
M A L E RECOMBINATION IN DROSOPHILA
315
B
A
R
cnbw 88 G,,
1 F4
R d cn bw
cn bw 89
FLY COUNTS EXPERIMENT 1
cn bw 98 G4
cn bw
I
FLY COUNTS EXPERIMENT 1
FIGURE 1.-A. Mating scheme utilized to isolate recombinant chromosome types (R) generated between the apl and T-007second chromosomes, including isolation of the presumptive nonrecombinant chromosomes of the genotype and to subsequently test them for the ability to induce male recombination between the second chromosome right arm markers cn and bw. B. Mating scheme utilized to isolate the presumably non-recombinant chromosomes of the a2 d p b pr c pz sp genotype, and to subsequently test them for the ability to induce male recombination between cn and bw.For full details consult text.
+ + + + + + +,
nant chromosomes which were generated in the heterozygous F, females. These recombinant chromosomes were then assayed for the presence or absence of the ability to induce male recombination between the two recessive markers cn and bw. This was done as follows: The F, males of the appropriate recombinant genotypes were individually mated to cn bw females, and from each of these matings, F, males were isolated. All F, males collected from each Fzmating were phenotypically “wild type” but genotypically either apl/cn bw or recombinant chromosome type/cn bw. It was therefore necessary to double-mate individual F, male
316
B. E. SLATKO A N D Y. HIRAIZUMI
progeny from each original F, recombinant male first to apZ females (FZain Figure 1( A ) ) to select males of the appropriate genotypes and then to cn bw females (Fa,,in Figure 1(A)) to isolate a larger number of recombinant chromosome type/cn bw males. The F4males were genotypically heterozygous f o r cn bw and the identical “recombinant chromosome” present in the F, parental males. These heterozygous males were then mated to cn bw females to test f o r the presence of the ability to induce male recombination. By this scheme, each of the individually isolated recombinant genotypes could be tested for the ability to induce male recombination between cn and bw, and in addition, it was possible to obtain a fairly large number of F, males (i.e., replications) for each individually isolated recombinant chromosome. It should be noted that from this mating scheme it is also possible to isolate the genotype presumptive non-recombinant chromosomes of the (= T-007),but impossible to isolate the non-recombinant apl chromosomes. This is due to the fact that in the F, matings in Figure 1(A), it is impossible to distinguish the apZ chromosome derived from the F, females and the apZ chromosome from F, males. For the purpose of isolating the non-recombinant apt chromosomes, an alternative mating scheme was used, which is shomwn in Figure 1 (B) . The results of these matings, designated as Experiment 1, are summarized in Table 1. Table 1 includes the results obtained for the 12recombinant and 2 presumptive non-recombinant genotypes (Type numbers 1-14) generated in the T-O07/apZ heterozygous females, which were tested for the ability to induce male recombi-
++ ++ +++
TABLE 1 Male recombination frequencies between the cn and bw loci for the twelve recombinant and two presumptive non-recombinant chromosome types generated in T-O07/apl heterozygous females TJ-w
Group number
A
1 2 3 4
B
5 6 7 8 9 10 11
c
12 13 14
N2+
Avg. no. replic. per line
Avg. no.
NI*
progeny per line
RE. h-eq. between cn and bw
9 6
0 0 0 0
19 4 9 6
28.5 31.5 21.3 32.2
1682 1727 1399 1874
0.0397 0.0100 0.0079 0.0398
aZdpbpr+++ a l d p b p r c + + a1 d p b p r c px a1 dp b p r c px sp dp b pr c px sp b pr c p x s p pr c P X SP
10 6 5 21 4 9 7
6 3 2 9 0 2 0
4 3 3 12 4 7 7
43.5 25.0 18.0 24.4 27.6
2235 1323 2199 1612 1330 1930 1585
0.0018 0.0005 0.0012 0.0009 0.0019 0.0020 0.0025
+ + + + + + S P
11 10 4
1 0 0
10 10 4
41.2 22.5 25.5
2187 1471 1393
0.0061 0.0063 0.0102
iTotal no. of lines isolated
Recombinant chromosome type
+++++++ al++++++ aldp+++++ a l d p b + + + +
19 4
+
+ ++ +++ f + + + c px s p $. + -k + + p x s p
19.2 44.0
~~
* Nupber of lines which did not induce any male recombination.
-f Number of lines which induced some male recombination. For further details, consult text.
MALE RECOMBINATION I N DROSOPHILA
317
nation between the second chromosome right arm markers cn and bw. For each recombinant chromosome type isolated (F, or Gzmales in Figure 1) ,a number of independently isolated chromosome lines were collected (designated as “Total number of lines isolated” in Table 1). For each independently isolated chromosome line, a number of replications were made (corresponding to the F4o r G4 males, Figure 1). The average number of replications and the average number of subsequent progeny scored for each chromosome line are shown for each recombinant chromosome genotype. Additionally, the number of independently isolated recombinant chromosome lines showing male recombination induction are listed, along with the recombination frequency (measured between cn and bw) averaged for all lines of each recombinant chromosome type. For example, in Group B, 21 independent (F,) males with the a l d p b p r c p x s p genotype (type number 8) were isolated. For each of the 21 lines, the number of replications (F, males) averaged 25.0 and the number of progeny (averaged for the 21 lines) was 1612. Of the 21 lines, 12 induced some male recombination. The recombination frequency between cn and bw, averaged for all 21 lines, was 0.0009 (almost 0.1 %). Because the phenomenon of male recombination seems to be of largely premeiotic origin (HIRAIZUMI et al. 1973), any computations of average recombination frequencies must take into consideration the occurrence of clustering. Although a deviation in the distribution of recombinants among males from the Poisson expectation was statistically significant, the degree of clustering was generally small. In addition there were no large differences, with respect to clustering, between the 14 recombinant chromosome types. For all practical purposes, therefore, the use of a simple average recombination frequency is permissible, neglecting the small clustering effect. The recombinant chromosome types listed in Table 1 have been organized into three groups (Groups A to C) . It can be seen that with respect to the ability to induce male recombination, the three groups can be classified into two sets. The first set (Groups A and C) induces a relatively higher frequency of male recombination than the second set (Group B) . The recombinant types in the first set (Groups A and C) have only one region in common: the pr+ region derived from the T-007 chromosome. Conversely, those recombinant chromosome types in the second set contain the pr region from the apl chromosome. Note that of a total of 63 chromosomes carrying the pr+ region which were tested, only one (of the type + + + + c p x s p ) did not induce any male recombination. All of these observations seem to suggest that one major element responsible for the induction of male recombination is located near, and probably to the right of, the pr+ locus in the T-007 chromosome. As was mentioned previously, practically all (i.e., 62 out of 63) of the recombinant chromosomes carrying the pr+ region from T-007 induced male recombination. If one assumes that only a single element is responsible for male recombination induction, the majority of the complementary recombinant chromosome types (which carry the pr region from the apl chromosome) should not induce male recombination. However, this does not appear to be the case; the
318
B. E. SLATKO A N D Y . HIRAIZUMI
majority (40 out of 58) show the induction of male recombination, although in markedly reduced frequencies. These results suggest that in addition to the major element located near the pr+ region of the T-007 chromosome, there must be at least one additional “secondary element” present which is also responsible for the induction of male recombination, If a single “secondary element” which can induce low levels of male recombination is assumed, then by examination of the lines in Group B (lines not carrying pr+ from T-007), it should be possible to’ localize this element. The assumption of a single second locus would necessarily imply the existence of two distinct classes of male recombination in this group. It might appear, by preliminary inspection, that a “secondary element” may be present around the al+ locus, since 18 out of the 20 recombinant chromosome types with al+ (types 9, IO, and 11) showed some male recombination. It can also be seen, however, that this single “secondary element” model does not entirely explain the observed results, since more than half (22 out of 42) of the remaining recombinant types (types 5 , 6, 7, and 8) also show some male recombination induction. One other interesting observation from the data in Table 1 that pertains to the analysis of the “secondary elements” concerns the presumptive non-recombinant chromosome of the genotype a1 d p b pr c p x sp. Flies of this type carry all seven mutant markers. and therefore have the same genotype as the standard apl chromosome, except f o r the possible occasional occurrence of double crossover events (in the T-O07/apl females) between any two adjacent markers. Also, possible single crossover events outside of the a1 and sp markers (to the left of a1 or to the right of sp) might occur such that the tip of the left or right arm of T-007 could be transferred to the “non-recombinant” chromosome. The frequency of such undetected double crossovers should be very low, as should be the frequency of recombination outside the two end markers, considering their map positions relative to the tips of the chromosomme arms. A total of 21 a1 d p b pr c px sp “nonrecombinant” chromosomes were tested for the ability to induce male recombination, as shown in Table 1. Of the 21 lines tested. 12 showed the ability to induce male recombination. This frequency (60%) is too high to be explained by the occurrence of double crossing over between any two adjacent markers, or by single crossover events outside the end markers (a1 and sp) . It should be stated here that neither the apl chromosome nor the cn bw chromosome induces any male recombination. From the mating apl/cn bw 8 X cn bw 0 , 7656 progeny were scored and no recombinants were observed. Similarly, from the mating apl/cn bw 8 x apl 0,4471 progeny were scored, and again, no recombinants were found. Thus, the c n bw chromosome and the apl chromosome do not contain genetic elements which can cause male recombination. These results suggest that in addition to the presence of the major locus f o r male recombination induction, ‘Lsecondaryelements” must be present which can induce low levels of male recombination. Since the T-007 second chromosomal line was originally kept by repeated backcross matings to cnbw, only the originally isolated second chromosome remains in the T-O07/Cy stock. Any genetic elements located on any other chromosome would have been diluted out.
319
M A L E R E C O M B I N A T I O N IN DROSOPHILA
Thus, there must be an association between the “secondary elements” and the T-007 second chromosome. We will return to reconsider these “secondary elements” later. Many of the chromosome lines included in Table 1 were discarded after the completion of Experiment 1. Several of the lines, however, were kept by repeated backcross matings through males to cn bw females. After six generations of these backcross matings, these lines were re-examined for the ability to induce male recombination. The results of these matings, designated as Experiment 2, are shown in Table 2. Experiment 2 was originally designed to obtain some confirmation of the results presented in Table 1. The results, however, provided much more important information than a mere confirmation. Whereas the data presented in Table 1 is a summary of all independently isolated chromosome lines of a given genoTABLE 2 Comparisons of male recombination frequencies for several recombinant chromosome types between Experiment 1 and Experiment 2 Experiment 1
Group
A
Recombinant chromosome type
+++++++ ++ ++ ++ ++ ++ ++ ++ +++++++ -
Line number
50 50 47 47
2421 2690 1964 2298
0.0050 3.0126 0.0041 0.0083
20 17 20 19
935 858 1087 926
0.0011 0.0163 0.0055 0.0088
al++++++ al++++++
3 4
48 43
2175 2388
0.0092 0.0101
14 20
739 1184
0.0149 0.0042
aldp b dp b
2
44 47
2109 2532
0.0079 0.0122
20 20
1271 1306
0.0047 0.0100
++ ++ ++ ++
5
0.0087
al d p b pr c
++
a1 d p b p r c px s p
a1 d p b pr c p x s p
a1 a1 a1 a1
-
dp dp dp dp
b b b b
0.0082
4
20
890
0.0000
20
1267
0.0000
8 9
17
20
942 980
0.0000 0.0000
20 20
1214 1333
0.3000 0.0000
o.cooo
Mean
c
Experiment 2
No. No. Rec. freq. males progeny between tested scored cn and bw
8 9 11 13
Mean
B
-
No. Rac. freq. No. F male: progeny between tested scored cn and bw
c px s p c px sp
0.0000
7 10 11 12
23 47 20 40
1007 2424 909 1749
0.0020 0.0017 0.0022 0.0017
30 30 29 30
2033 2093 1741 2102
0.0000 0.0000 0.0000 0.0000
3 4
23 20
942 920
0.032 0.0011
40
44
2694 2410
0.0000
p r c P X SP
P r c PZ S P
3
48
2275
0.0304
40
2874
0.0000
pr pr pr pr
+ dP b ‘P + 4~b +++
c px s p c px s p
c
PZ SP
Mean
For full details, consult text.
0.0018
0,0000
0.0000
320
B. E. SLATKO A N D Y. H I R A I Z U M I
type, the data in Table 2 is presented so that for each individual line of a given genotype, direct comparisons can be made between the results oibtained in Experiment 2 (after 6 generations of backcross matings) . Before examining the results presented in Table 2, one aspect which first should be considered concerns any change in the “recombinant chromosome genotype” due to male recombination which could have occurred during the s i x generations of backcross matings to the cn bw females. Any recombination between the cn and the bw loci could be detected by phenotype. In fact, such recombinants were occasionally found and eliminated during the stock-keeping procedures. Since the cn bw chromosome does not carry any markers in the left arm, male recombination in the left arm could not be detected in this manner. Before experiment 2 was performed, a check was made for the presence of the left arm markers characteristic for each of the “recombinant chromosome” lines. This screening was done by mating the heterozygous males, (recombinant chromosome type /en bw), to apl females and examining the phenotypes of the progeny flies. This works efficiently for the lines carry the left arm end marker, al, but does not work for lines such as c px sp, which lack left arm markers. Consequently, there is the small possibility of undetected male recombination in the left a n n of such genotypes. In addition, there is no way to detect any double crossover events which might have occurred between any two adjacent markers, or single crossover events outside of the two end markers, al, and sp. The possibility of such undetected male recombination should be very small, however, since the frequency of male recombination is generally low. Based upon these considerations, it seems probable that a large majority (presumably all) of the “recombinant chromosome” lines tested in Experiment 2 still maintained the same genotypes originally present when they were tested in Experiment 1. In Table 2, the recombinant chromosome types are divided into three groups (Groups A, B, and C) . Group A includes those lines which carry the pr’f region from T-007 and which induced relatively higher frequencies of male recombination in Experiment 1 (Mean = 0.0087). When retested after six generations of backcross matings, these lines continued to induce male recombination in comparable frequencies (Mean = 0.0082). As mentioned previously, several lines tested in Experiment 1 failed to induce male recombination. With only one exception, these were restricted to lines which carry the pr region from the apl chromosome. Group B in Table 2 lists such lines, i.e., those lines which induced no male recombination in Experiment 1 and which were retested in Experiment 2. It can be seen that those lines which were unable to induce male recombination in Experiment 1 remained unable to do so when retested after the six backcross generations. The most interesting observation from the date presented in Table 2 concerns those lines which are inchded in Group C. These are the lines which carry the pr region from the up2 chromosome and which showed relatively lower frequencies of male recombination in Experiment 1. When these lines were retested after six generations of backcross matings, none was able to induce male recombination any longer. In these genotypes, the ability to induce male recombination is now very much reduced.
+ +++
321
MALE RECOMBINATION I N DROSOPHILA
Because there was a previous suggestion that the “major element” responsible for the induction of male recombination locates between the pr+ and c+ loci in the T-007 chromosome, the two complementary recombinant chromosome types generated between these two markers (i.e., c px s p and a2 d p b p r +) were examined separately from the recombinant types listed in Table 2. The results from the matings designed to retest these two recombinant types for the ability to induce male recombination after the six generations of backcross matings are shown in Table 3. Table 3 is analogous to Table 2 (in design and intention), with the exception that only the two complementary recombinant chromosome types, c p x sp and a2 d p b pr are considered. Group A includes those lines which induced relatively higher frequencies of male recombination in Experiment 1. It can be seen that these lines have retained the ability TO induce male recombination when retested after six generations of backcross matings. It is interesting to note that both of the complementary genotypes are represented in this group, as expected if the “major element” locates between the p i + and c+ regions of the T-007 chromosome. Group B in Table 3 includes those lines which induced no recombination in Experiment 1. The lines included in this group are still unable to induce male recombination, analogous to the data presented for Group B in Table 2. Group C in Table 3 contains a single representative. This line induced relatively lower male recombination in Experiment 1; but when retested after the 6 generations of backcross matings, this line was found to be unable to induce
+ +++
++
++++
+ + +,
TABLE 3
+++
Comparisons of male recombination frequencies for the a1 dp b pr and px sp recombinant chromosome types between Experiment I and Experiment 2
+ + + +c
~
~
~~~
Expenment 1
No F4 Group
A
Recombinant chromosome type
++++ ++ ++ ++ ++ a1 dp b pr a1 dP b Pr
c PXSP c PXSP
c PZSP
++ ++ ++
Line number
No.
Rec. freq. males progeny between tested scored cn and bw
~-
5 6 7
50 37 47
2266 2491 2975
4 10
20 53
1960 2716
++ ++ ++ a l d p b p r + + + al dP b Pr + + + dp b pr
2 3 8 9
45 50 40 40
2104 2328 2630 2390
a l d p b p r + + +
For full details, consult text.
1
21
1900
Dec. f q .
0.006.E
20 20
1063 1336 1348
0.0019 0.0112 0.0052
0.0034 0.0056
40 30
2557 1972
0.0023 0.0076
0.0013 0.3129
20
0.0000 0.0000 0.0000 0.0000
0.0056 30 20 20
20
1819 1142 1330 1477
0.0000
Mean
c
No.
males progeny between tested scored cn and bw
0.0059
Mean al dP b Pr
Experiment 2
No.
0.0046
0.0000 0.0000 0.0300 0.0000
0.0000 30
1660
0.0000
322
B. E. SLATKO A N D Y. H I R A I Z U M I
male recombination any longer. This line thus acts similarly to those lines included in Group C in Table 2. These results seem to indicate that the maintenance of the ability to induce male recombination is due to a single element located between pr+ and C+ in the T-007 chromosome, as some of the lines of the genotype a1 d p b pr 4-4- still maintain the ability to induce male recombination in Experiment 2, and thus still carry the “major element”. This element may locate nearer to pr+ in the T-007 chromosome, although the number of lines tested is too few to determine its position accurately. When a chromosome carries this element, to which we give the name Mr (for Male recombination), this genotype has the ability to exhibit similar levels of male recombination through successive generations. AS was pointed out previously, M r is not the only element; there seem to be other “secondary elements” present which can induce male recombination in much reduced frequencies. Results of Experiment 2 indicate that the action of such “secondary elements” is rather transient, inducing male recombination f o r only a few generations.
+
DISCUSSION
The male recombination system in Drosophila melanogaster appears to be a complex system in which one major element, Mr, is responsible for the large majority of male recombination. Mr locates between the p r f and c+ loci in the T-007 chromosome, possibly nearer to the pr+ locus. In addition to this major element, there seem to be other “secondary elements” present whose abilities to induce male recombination are much less than that of Mr and whose effects are “diluted O U ~ ” through successive backcross generations. It is obvious that the elements responsible for male recombination must be associated with the second chromosome. However, this does not preclude the fact that this strain of Drosophila melanogaster could have had elements located on other chromosomes. Because the original isolation selected only the second chromosome, the influence of the other chromosomes is unknown. In fact, VOELKER (1974) and WADDLE and OSTER(1974) have obtained results that indicate that when both second and third chromosomes are extracted simultaneously from males isolated from “natural populations”, a higher frequency of male recombination is observed than if just the original second chromosome or the original third chromosome is present. At the present time, little data is available concerning the nature of the transitory “secondary element” effect. It was mentioned previously that neither the apl chromosome nor the cn bw chromosome induces male recombination (apl/cn bw males x cn bw females-7656 progeny, 0 recombinants; upl/cn bw males x up1 females-471 progeny, 0 recombinants). Thus, it is unlikely that either one of these stocks carries M r or any “secondary elements” that can induce male recombination. It is possible to envision that during backcrossing to c n bw,modifiers (which elements”) from the apl stock were being could be interpreted as LLsecondary diluted out. This would imply, however, that the apl stock must behave differ-
MALE RECOMBINATION IN DROSOPHILA
323
ently than the cn bw stack. Neither of these stocks induces male recombination, nor does either of these stocks show any type of suppression o r enhancement phenomenon. It is reasonable to conclude that the loss of “modifiers” (originally present in the apl stock) or the gain of “suppressors” (originally present in the cn bw stock) are not likely explanations for the evanescent “secondary element” effect. Ruling out these possibilities, the simplest explanation for this phenomenon rests with the T-007 genotype itself. It is interesting to note that this temporal “secondary element” effect could be the explanation for the speculations concerning transposable elements (HIRAIZUMI et al. 1973; KIDWELLand KIDWELL 1973; VOELKER 1974; WADDLE and OSTER1974) that have been advanced in order to explain some unusual aspects of this system. A number of other possible speculations concerning the transitory “secondary element” effect can be made. This phenomenon could perhaps most easily be explained by assuming that the “secondary elements” are products of M r ; however, one must consider the fact that the temporal effects are manifest for several generations. Obviously, a simple protein product of M r is an unlikely candidate. In fact, there is no a priori reason to assume that the “secondary elements” are physical particles. M r may induce some change in its homolog, which is temporal. This is similar in a number or respects to the paramutation phenomenon seen in a number of organisms (for recent review, see BRINK1973) and/or ribosomal DNA “magnification” seen in Drosophila melanogaster (RITOSSA 1968; TARTOF 1974). It is our feeling that a number of the previously mentioned speculations are experimentally testable. Investigations into this aspect of the male recombination system are the subjects for future studies. LITERATURE CITED
BRINK,R. A., 1973 Paramutation. Ann. Rev. Genet. 7: 129-150. BROADWATER, C., L. V. OWENS,R. PARKS,C. WINFREY and F. R. WADDLE,1973 Male recombination from natural populations of Drosophila melanogaster from North Carolina. Drosophila Inform. Serv. 50: 99. FRANCA, Z. M., A. B. DA CUNHA and M. C. GARRIDO, 1968 Recombination in Drosophila willisloni. Heredity 23: 199-204. HINTON,C. W., 1970 Identification of two loci controlling crossing over in males of Drosophila ananassae. Genetics 66:663-676. HIRAIZUMI, Y., 1371 Spontaneous recombination in Drosophila melanogaster males. Proc. Natl. Acad. Sci. US. 68:268-270. HIRAIZUMI, Y., B.SLATKO, C. LANGLEY and A. NILL, 1973 Recombination in Drosophila melanogaster male. Genetics 73 : 439-444.
M. G. and J. F. KIDWELL, 1975 Cytoplasm-chromosome interactions in Drosophila KIDWELL, melanogaster. Nature 235 : 755-756. KIKKAWA, H., 1937 Spontaneous crossing over in the male of Drosophila ananassae. 2301. Mag. 49: 159-160.
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MORGAN, T. H., 1912 Complete linkage in the second chromosome of the male of Drosophila. 1914 No crossing over in the males of Drosophila of genes in Science 36: 710-720. -, the second and third pairs of chromosomes. Biol. Bull. 26: 195-204. MORIWAKI, D. and Y. N. TOBARI, 1973 Spontaneous male crossing-over of frequent occurrence in Drosophila ananassue from southeast Asian populations. Japan. J. Genetics 48: 167-173. RITOSSA,F., 1968 Unstable redundancy of genes for ribosomal RNA. Proc. Natl. Acad. Sci. U.S. 60: 509412. SLATKO,B. and Y. HIRAIZUMI, 1973 Mutation induction in the male recombination strains of Drosophila melanogaster. Genetics 75 : 643-649. SVED,J. A., 1974 Association between male recombination and rapid mutational changes in Drosophila mehanogaster. Genetics 77 (suppl.) : 64. TARTOF, K. D., 1974 Unequal mitotic sister chromatid exchange and disproportionate replication as mechanisms regulating ribosomal RNA gene redundancy. Cold Spring Harbor Symp. Quant. Biol. 38: 491-500. VOELKER, R. A., 1974 The genetic and cytology of a mutator factor in Drosophila melanogaster. Mutation Res. 22 : 265-276.
WADDLE, F. R. and I. I. OSTER,1974 Autosomal recombination in males of Drosophila mehnogaster caused by a transmissible factor. J. Genet. 61: 177-183. YAMAGUCHI, 0. and T. MUKAI,1974 Variation OF spontaneous occurrence rates of chromosomal aberrations in the second chromosomes of Drosophila melanogaster. Genetics 78: 1209-1221. Corresponding editor: A. CHOVNICK
